Although human immunodeficiency virus type 1 (HIV-1) is clearly the etiologic agent of AIDS, certain, as yet undefined cofactors likely play an important role in HIV-1-related disease progression to AIDS. Human cytomegalovirus (HCMV) is a major pathogen in HIV-infected individuals and infects many of the same cells as HIV-1. During the first 4.5 years of this grant, we have elucidated the complex interactions between HIV-1 and HCMV in brain-derived cell lines and in macrophages. Our studies indicate that multiplicity of infection, the relative permissiveness of the cell for the two viruses, and the production of cytokines can affect the balance between stimulation or inhibition. We have also shown that the response of the HIV-1 promoter to HIV-1 and HCMV regulatory proteins is dependent on whether the DNA is integrated and that transient expression assays with transfected constructs do not accurately reflect the molecular interactions occurring when the HIV-1 genome is integrated into the host genome. The Specific Aims of this proposal are: (1) To analyze the effect of dual infection with HIV-1 and HCMV in the highly relevant systems of monocyte-derived-macrophages, long-term bone marrow cultures, and primary cultures of brain cells. We will determine: What cell types are involved in the co-infection? Do HIV-1 and HCMV replicate in the same cell? If not, and there is modulation of the HIV-1 or HCMV infection, is it occurring through the production of soluble factors? What are the patterns of viral DNA replication, RNA transcription, protein synthesis, and virion production? (2) To characterize the molecular basis of the stimulatory and inhibitory effects of HCMV and HIV-1 transcription. Cell lines containing an integrated HIV-1 promoter and HIV-1 genes driven by an inducible heterologous promoter (the Stratagene Lac Switch system) will be used to identify the cis-acting sequences in the HIV_1 LTR, and the HIV-1 and HCMV gene products required for the HCMV-mediated effects; and (3) To determine if HCMV infection hastens both the qualitative loss of T-cell function and eventual T-cell death through apoptosis associated with HIV-1 infection. Accomplishment of the above goals will provide an important foundation for understanding the role of viral cofactors in AIDS, and establish the basis for development of effective approaches for treating and preventing HIV- related disease.